Diaminooxidase-Containing Pharmaceutical Compositions

ABSTRACT

The present invention relates to pharmaceutical compositions, food supplement compositions and cosmetic compositions comprising diaminooxidase, and to the use thereof.

The present invention relates to compositions for the treatment and/orprevention of histamine-induced diseases and conditions.

Histamine (1H-imidazole-4-ethylamine) forms by the enzymaticdecarboxylation of histidine and, thus, is a basic biogenic amine havinga molecular weight of 111 Da.

In the organism, histamine occurs practically ubiquitously. It isproduced by humans themselves and stored in inactive form in themetachromatic granules of the mast cells and basophilic leukocytes,where it is available for an immediate release. The highest histamineconcentrations are measured in the lungs. After its release, histamineis a highly potent mediator for a plurality of physiological andpathophysiological processes, often also via an interaction withcytokines.

Moreover, histamine may also get into the body from the outside, byinhaling, on the one hand, or orally, e.g. by ingestinghistamine-containing foodstuffs, such as cheese, wine, canned fish andsauerkraut.

The most important functions and effects of histamine in the human andanimal body, respectively, are:

1) Dilation of the capillaries, increase in the capillary permeabilityand drop of blood pressure.

2) Contractions of the smooth muscles, i.a. of the bronchial muscles inthe lungs.

3) Induction of an increase gastric acid secretion.

4) Increase in the heart rate.

5) Histamine is the mediator of the allergic immediate type reaction, itis the most important mediator in allergic diseases, such as Rhinitisallergica (hay fever) and Asthma bronchiale.

6) Moreover, histamine is the classical trigger of an urticaria (nettlerash) and it plays an important role in drug allergies or intolerances.

High concentrations of freely circulating histamine initiate undesiredeffects, such as headaches, stuffy or runny nose, obstruction of therespiratory tract, tachycardia as well as extrasystoles, andfurthermore, gastro-intestinal complaints which may lead from loosestools up to diarrhea, and hypotension. Often also swellings of theeyelids, sometimes also urticarial exanthemas have been described.Moreover, reddening of the skin, a drop in blood pressure andbronchospasms may occur. The following table shows the symptoms as afunction of the histamine concentration in blood.

Histamine (ng/ml) Body reactions 0-1 none 1-2 Increase gastric juicesecretion 3-5 Tachycardia, skin irritations 6-8 Drop in blood pressure 7-12 Bronchospasm approx.100 Cardiac arrest

In the organism of mammals, histamine is degraded by two enzymes:diaminoxidase (DAO, EC 1.4.3.6) and histamine-N-methyltransferase (NMT,EC 2.1.1.8) (Mizugushi et al., 1994). DAO catalyzes the oxidativedeamination of histamine to imidazole acetaldehyde; NMT catalyzes theN-methylation to N-methyl-histamine.

Both degradation pathways are essential to the organism: DAO removeshistamine which has, e.g., been taken up in the gastro-intestinal tractvia the food, NMT controls the histaminergic signal transmission in thenervous system (Kitanaka et al., 2002).

It is the main objective of DAO to prevent histamine that has beeningested with the food from getting from the intestines into the bloodcirculation. Failure of this protective mechanism may result inanaphylactic shock in extreme cases (Taylor 1986, Nilsson et al., 1996).DAO is a secretory protein and, thus, acts extracellularily, whereasN-methyl-transferase is exclusively active in the cytosol (Küfner etal., 2001).

Native DAO which can degrade further biogenic amines, such as, e.g.,putrescin, spermidin and cadaverine, in addition to histamine, and whichmay, e.g. be recovered from porcine kidney, is a homodimericcopper-containing glycoprotein, in which the sub-units are connected viadisulfide bonds. DAO has a molecular weight of approximately 182 kDa(Kluetz and Schmidt, 1997; Rinaldi et al., 1982) and a carbohydrateportion of approximately 11% (Shah and Ali, 1988). The enzyme belongs tothe class of the copper-containing aminooxidases, which catalyze theoxidative deamination of primary amines to aldehydes, ammonia andhydrogen peroxide according to the following general reaction scheme(Bachrach 1985): RCH₂NH₂+H₂O+O₂=>RCHO+NH₃+H₂O₂, the residue R containingan amino group.

Characteristic of the copper-containing amino oxidases is a topaquinoneat the active center which forms by post-translational modification of aconserved tyrosine-residue (James et al., 1990, James et al., 1992; Muet al., 1992).

DAO is mainly found in the small intestines, in the liver, in thekidneys and in blood in white blood cells. In pregnant subjects, DAO isadditionally formed in the placenta. Pregnant subjects have a blood-DAOlevel that is higher by approximately 500 to 1000 times than that ofnon-pregnant subjects. DAO is continuously produced and excreted intothe intestinal lumen. In healthy humans, food rich in histaminestherefore is already largely freed from histamine in the intestines. Theremaining histamine is degraded when it passes through the intestinalmucosa by the DAO present there. Histamine is decomposed toimidazole-acetaldehyde and, furthermore, to imidazole-acetoacetic acid.The cofactors of DAO are 6-hydroxydopa and presumablypyridoxalphosphate, the vitamin B6. DAO is a sensitive enzyme which canbe inhibited by various substances, i.a. by biogenic amines, alcohol andits degradation product acetaldehyde, and various medicaments. Inneuronal tissue, so far no DAO activity could be detected.

As has already been mentioned before, exogenous histamine ingested withthe food, but also endogenous histamine can trigger a large variety ofdisorders due to allergic reactions. With regard to its clinical value,at least three forms of a histamine intolerance based on a reduced DAOactivity are to be pointed out:

-   -   Few people have a congenital DAO deficiency and do not lose it,        either.    -   During an infection of the intestinal mucosa, a transient DAO        deficiency may occur. When the infection has been healed, also        the DAO activity returns to normal.    -   When administering various activity-inhibiting substances, there        may result exogenously a reduced DAO activity. Among them are        primarily alcohol and its degradation product, acetaldehyde,        certain foodstuffs rich in amines, and many medicaments.

In all the cases, the symptoms initially described occur more or lessstrongly and cannot be attributed easily in most instances. A rapidclarification of the functional activity of the enzyme allows for arapid and simple therapy and for providing an appropriate dietary plan.

A frequent reason for the occurrence of a histamine intolerance is thesensitivity of the enzyme relative to a large number of chemicalsubstances. Many of them occur in different medicaments. The mostimportant DAO inhibitors are acriflavine, diazepam,N-methyl-N-formylhydrazine, b-aminopropionitriles, Dimaprit,O-methylhdroxylamine, agmantine, ethanol (10%), Pargyline, Aldomet,Furosemide, Phenamil, Amiloride, Guanabenz, Phenelzine, Aminoguanidine,Guanfacine, Phenformin, Amitryptiline, guanidine, phenyprazine,amodiaquine, haloperidol, promethiazine, anserine, Hyamine 1622,propranolol, aziridinyl-alkyl-amines, hydroxychloroquines, B1pyrimidine, hydroxylamine, quinacrine, Burimamide, Impromidine,semicarbazide, cadaverine, imidazol derivatives, thiamines, Carnosine,iproniazid, thioridazine, chlorothiazide, isocarboxazide,tranylcypramine, chlorpromazine, isoniazide, trimethoprim, cimetidine,metiamide, tryptamine, clonidine, metronidazole, tyramine, cyanide,nazlinin (alkaloid), diamines (also histamine) and Nt-methyl-histamine.

In WO 02/43745 the systemic use of DAO of plant origin for the treatmentof histamine-mediate diseases is disclosed. The administration of DAO orof enzymes in general which are directly isolated from plants is a greatproblem because of the frequent occurrence of allergens in plants,primarily in view of the fact that the leguminous plants disclosed in WO02/43745 have a high allergenic potential.

It is an object of the present invention to provide compositions for thetreatment and prevention of histamine-induced diseases and conditionswhich do not have the side-effects of products available on the marketwhich mainly comprise cortisone, and which eliminate the disadvantagesencountered in the prior art mentioned above. It is a further object ofthe present invention to increase the concentration of activediaminooxidase within the body, in particular in the intestinal tract,of an individual to thereby assist in, or enable, respectively, thedegradation particularly of histamine that has been exogenicallysupplied (e.g. with the food).

Therefore, the present invention relates to pharmaceutical compositionsfor the treatment of histamine-induced diseases, comprisingdiaminooxidase, wherein the composition is provided in an applicationform for epidermal, oral, peroral or sublingual administration as ahydrogel, gastric-juice-resistant pellet, drops, in particular eye dropsand nose drops, or as a tablet, food supplement compositions, or dietaryfoodstuffs and cosmetic compositions comprising diaminooxidase. In thecompositions according to the invention, the diaminooxidase is providedsubstantially in its active form, which means that enzymes which, forinstance, do not comprise copper in their prosthetic group and do not atleast exhibit wild-type activity, are not suitable for the applicationsaccording to the invention.

The pharmaceutical composition according to the invention comprises apharmaceutical form which allows for an administration selected from thegroup consisting of epidermal, oral, peroral and sublingualadministration. The epidermal administration of DAO is primarilyadvantageous in case of histamine-induced diseases, or conditions,respectively, at the surface of the skin, or on the outermost layers ofthe skin, respectively, since by this it is, e.g., possible tosuccessfully treat allergic reactions that resulted from a contact ofthe skin with an allergen. Likewise itching, caused in various diseases,like urticaria, atopic eczema and the like, triggered by a release ofhistamine, can be stopped. By the oral and peroral administration of thecompositions according to the invention it is possible to get DAO intothe gastro-intestinal tract of an individual and to successfully stop,or treat, respectively, the histamine-induced illnesses there bydegrading histamine. The range of activity of the DAO is mainlyrestricted to the gastro-intestinal tract since the high acid content inthe stomach has a negative effect on the activity of DAO. Therefore,when administering DAO orally or perorally, it is necessary to protectDAO from gastric acid until it has reached the intestinal tract.However, if DAO is administered sublingually, the enzyme is quicklytaken up in the mouth by the oral mucosa and delivered into thebloodstream. In this way it is possible to quickly and easily transferDAO into the bloodstream without having to trans-port the enzymeintravenously or having to transport it through the stomach to theintestines without any harm, which delays a rapid start of the enzymeeffect.

The pharmaceutical composition is provided in a form of administrationselected from the group consisting of hydrogel, gastric-juice-resistantpellet, drops, in particular eye drops and nose drops, tablets andcapsules. According to the present invention, it is possible to convertDAO by processing methods according to those known in the prior art intopharmaceutical forms to be administered. Therefore, pharmaceuticalcompositions comprising DAO also contain further ingredients which areused to stabilize the enzyme, on the one hand, and to bring the enzymeinto the corresponding galenic form, on the other hand. DAO may, ofcourse, also be administered together with other pharmaceutically activesubstances in a single form of administration, or separately, as long asthe enzyme is not inhibited by any one of these active substances suchthat an activity of the enzyme does not unfold its desired effect.

The DAO comprising hydrogel compositions according to the inventionpreferably have a viscosity of from 0.5 to 5 cp (centipoise), morepreferably from 1 to 2 cp, in particular 1.1 to 1.6 cp, at a shearingspeed of 41 sec⁻¹, wherein the measurement may be effected by means ofviscosimeters known in the prior art. It has been found that theDAO-containing hydrogel composition has advantageous properties (e.g.topic distributing ability, good handling) particularly in theseviscosity ranges. At a lower viscosity of 0.5 cp, the hydrogelcomposition proved to be too thin in order to allow for a user-friendlyhandling of the preparation. Preferably, polyacrylates (e.g. Carbopol),cellulose derivatives, or modified cellulose, respectively, inparticular hydroxy ethylcellulose (e.g. Natrosol), methylcellulose,hydroxypropylmethyl cellulose and hydroxymethylcellulose, starch andmodified starch, natural and synthetic rubbers, such as, e.g.,tragacanth, guar, Carrageenan, gelatine, sodium alginate, PVP, polyvinylalcohol and mixtures thereof are used as gelatinizing agents. Asgelatinizing agent, particularly preferably, hydroxyethyl cellulose(e.g. Natrosol) is used, wherein, in particular, Natrosol Type 250 HHRexhibits a particularly good stability already at a low concentration.The pH of the hydrogel composition preferably ranges from 7.0 to 9.0,preferably from 7.2 to 8.5, more preferably from 7.5 to 8.0. This pHrange is preferably adjusted with a buffer, in particular with aBis/Tris buffer, wherein further salts (e.g. 20-300 mM, preferably from50 to 200 mM, in particular 100 mM, NaCl) may be added for maintainingthe DAO enzyme activity and stability. If the total concentration of thesalts is below 20 mM, the DAO has a poorer stability which may lead to aloss of activity.

Furthermore, also preservatives may be added to the hydrogel compositionaccording to the invention, which preservatives substantially preventthe proliferation of microorganisms in the composition. What isimportant here is to choose the preservatives such that theantimicrobial substances do not inhibit the DAO activity in a mannerthat its enzyme activity does no longer suffice to achieve the object ofthe present invention (degradation of histamine). By activity testswhich are carried out with DAO and the preservative, such substances canbe identified. Preferably, chlorhexidin, parabens (para-hydroxy-benzoicacid esters, in particular butyl, ethyl, methyl or propyl parabens),benzoates (e.g. Na-benzoate), sorbates (e.g. K-sorbate) carbamates (e.g.iodopropynyl butyl carbamate) and combinations thereof (e.g.Rokosonal=mixture of Na-benzoate, K-sorbate andiodopropynyl-butyl-carbamate) are used as preservative. Particularlypreferred are hydrogel compositions which comprise Natrosol asgelatinizing agent and Rokosonal and/or parabens as preservatives. Ofcourse, according to the invention also scents and aromatic substanceswhich are employed in the cosmetics industry, e.g., can be added.

A further aspect of the present invention relates to a food supplementcomposition, or to a dietary foodstuff (DFS) which is adapted as agastric-juice-resistant pellet, drops or infusion. The oraladministration of DAO by means of a food, a food supplement, or adietary foodstuff requires this enzyme to get into that part of the bodyin which it is to unfold its activity. Since histamine plays animportant role in the intestinal tract, it is necessary for the DAO tobe adapted or provided in a manner that it can pass the highlyacid-containing stomach unharmed. In this instance, the DAO preferablyis processed to a gastric-acid-resistant pellet.

Since at a pH of below 3, the DAO is irreversibly damaged (the pH in thestomach ranges between pH 2 and 4), in order to transport the DAOthrough the stomach into the intestinal tract it is necessary for theDAO to be provided in an appropriate form of administration(pharmaceutical composition or food supplement composition, or dietaryfoodstuff, respectively). According to the invention, capsules (e.g.gelatine capsules) and, in particular, gastric juice-resistant pelletshave proven particularly advantageous. It is an advantage if theactivity of the DAO starts 15 minutes at the latest, preferably 20minutes at the latest, in particular 30 minutes at the latest, afterhaving been administered.

According to the invention, “gastric-acid-resistant” is to denote thatproperty of the pellet which is capable of protecting an activesubstance (e.g. DAO) contained therein under the action of a gastricjuice, or of a solution having properties comparable to those of gastricjuice (e.g. acid) for a certain period of time of at least 10,preferably at least 20, more preferably at least 30, in particular atleast 60 minutes such that the active substance undergoes a loss ofactivity of 50% at the most, preferably 40% at the most, more preferably30% at the most, most preferred 20% at the most, in particular 10% atthe most.

Preferably, after 20 minutes, in particular after 30 minutes, the pelletaccording to the invention releases in the intestines at least 60%, inparticular at least 80% of the DAO activity which was used to formulatethe pellets.

Gastric-juice-resistant pellets are pellets which are coated by agastric-juice-resistant coating, which dissolves at a pH as is found inthe intestinal tract. This means that such coatings preferably dissolveat a pH of 4 at the least and 10 at the most. Eudragit, e.g., agastric-juice-resistant coating based on anionic polymers of methacrylicacid and methacrylates, contains —COOH as functional group and dissolvesin the range of pH 5.5 to pH 7. As an alternative to Eudragit, shellacor acetylated starch (e.g. Amprac 01) may be employed. Since thegastric-juice-resistant coatings known in the prior art have differentproperties (e.g. pH, at which the coating dissolves, dissolution rate),the materials of the coatings can also be combined. Shellac, e.g.,exhibits a good acid resistance, yet it dissolves very slowly in theintestinal tract. Amprac 01, on the other hand, dissolves rapidly in theintestinal environment, yet it is not sufficiently acid-resistant. Inorder to compensate the disadvantages of a material, the twoabove-mentioned materials may, e.g., be mixed at a weight ratio of60-95/40-5, preferably of 70-90/30-10, shellac/Amprac 01. A furtherparameter which has an influence on the release rate of the activesubstance is the layer thickness of the gastric-juice-resistant pellet.The layer thickness, expressed as mass ratio, is preferably 5 to 30%,more preferably 10 to 20%, of the total mass of the final product. Thepellets preferably have an average diameter of from 0.5 to 5 mm, inparticular of from 0.7 to 2 mm. Such a size has the advantage that thepellets can quickly pass the stomach.

Preparation of the Pellets of the Invention which may be used both inthe inventive pharmaceutical composition and in the inventive foodsupplement composition, or dietary foodstuff, respectively, preferablyis effected by means of an extruder which requires a thermal stabilityof the ingredients of the composition, in particular of the activesubstance DAO, of up to 60° C. (Stricker Arzneiformenentwicklung,Springer Verlag 2003). The pellets may comprise additionalpharmaceutical additives in addition to a gastric-juice-resistantcoating and DAO. For instance, microcrystalline cellulose (Avicel, e.g.)serves as a filler and swelling agent. Cellulose is insoluble in water,and in this form has both crystalline and also amorphous portions. Thiscombination causes a plastic deformability, which means that at asufficiently high force, an irreversible change in shape will occur.This is a substantial prerequisite for pelletizing in an extruder andspheronizer. During the moist granulation, the microcrystallinecellulose absorbs large amounts of water and by this becomes a readilycompressible, coherent mass, also without addition of a binder.According to the invention, the amount of microcrystalline cellulose ina pellet may range between 5 and 70%, preferably between 10 and 60%,even more preferred between 15 and 50%. As the binder or filler, sucrosemay be used. Sucrose increases the solubility of the matrix and thusassists in the rapid release of the enzyme. According to the invention,sucrose may be added to a pellet in an amount from 1 to 40%, preferably5 to 35%, even more preferably 10 to 30%. Hydroxypropyl cellulose(admixed in an amount of preferably 0.5 to 10%) can also be added as abinder and serves for preventing fine dust. Moreover, hydroxypropylcellulose increases the strength of the pellet and, thus, againcontributes to improving the yield. Corn starch can be added to thepellet according to the invention as a filler and disintegrating agent(in a preferred amount of from 1 to 30%). Being a water-insolublesubstance, starch can absorb a lot of water and, thus, is an idealdisintegrating agent. Crosscarmellose (Na-CMC; Acdisol) is a puredisintegrating agent which, preferably, can be used in an amount ofbetween 1% and 5%. Too high a portion of Acdisol will lead to an earlydisintegration of the pellet already during rounding thereof and, thus,is counterproductive. Crosspovidon, a cross-linked PVP, likewise iswater-insoluble and also serves as a disintegrating agent. Due to itspolymeric properties, it assists in an improved rounding during theproduction of pellets (may preferably be admixed in an amount of from0.5 to 10%). Povidon is a water-soluble additive and serves as a binder.The combination of these different fillers, disintegrating agents andbinders leads to a molecular-disperse distribution of the DAO in thepellet and ensures a rapid bioavailability.

Between the gastric-juice-resistant coating and the pellet with theactive agent, an insulating layer made of glycerol and/or talcum may beprovided. Glycerol serves as a humectant so as to prevent adehydrogenation and, thus, inactivation of the enzyme.

As an alternative to pellets, the DAO may also be transported incapsules through the stomach into the intestinal tract. Suitablecapsules are, e.g., gelatine capsules or starch capsules. The capsulesmay also contain the pellets according to the invention.

A further aspect of the present invention relates to a cosmeticcomposition comprising diaminooxidase, which is provided in a cosmeticadministration form, in particular as a hydrogel, ointment, spray or asdrops. In case of an increased histamine release, or in case of acontact with allergenic substances (e.g. in case of contact allergies orneurodermatitis) body reactions may occur at visible sites of the body,which reactions can be suppressed by administering DAO in cosmeticcompositions. Cosmetic compositions comprising DAO may, furthermore,comprise other ingredients known in the prior art, which are used in thepreparation of cosmetic products. Hydrogel-comprising cosmeticcompositions have substantially the same properties and containsubstantially the same ingredients as hydrogels of a pharmaceuticalcomposition according to the invention.

Preferably, the diaminooxidase used in the compositions according to theinvention is of non-plant origin.

The use of DAO of non-plant origin in pharmaceutical and cosmeticcompositions as well as in food supplements and in dietary foodstuffshas the advantage that allergens occurring in plants will not negativelyaffect the administration of DAO, since allergens substantially promotethe endogenous histamine release. It has been known that primarily plantsubstances are responsible for histamine-induced diseases. The completeremoval of allergy-triggering ingredients from a DAO preparation ofplant origin is possible only with a high preparative effort, whereasthe DAO according to the invention which is of non-plant origin iscompletely free from such plant allergens.

According to the present invention, by “non-plant origin” all the DAOsare comprised which are not recovered from plants, but from animalorganisms or from other non-plant organisms. Moreover, according to theinvention this definition includes all the DAOs which are recombinantlyprepared in cell cultures (animal, bacterial, yeasts and the like), orin non-plant organisms of any type, wherein the DNA for therecombinantly prepared DAO is isolated from plant and/or animalorganisms by methods known in the prior art, and cloned and expressed inexpression systems.

Preferably, all the compositions disclosed in the present inventioncomprise diaminooxidase of animal origin. By using animal DAO, it ispossible to provide the human, or animal body, respectively, with anenzyme which is very similar to the enzyme produced by these individualsthemselves in terms of glycosylation, activity and specificity of theDAO produced by these individuals themselves. Moreover, it is possibleto entirely exclude plant allergens from the production of DAO.

Preferably, the diaminooxidase is recovered from porcine kidneys.Porcine kidneys are primarily characterized by their high content ofDAO. From porcine kidneys, the enzyme can be isolated in a simple mannerby methods known in the prior art.

According to a further preferred embodiment, the compositions accordingto the invention comprise diaminooxidase of recombinant origin. By therecombinant production of DAO it is possible to produce large amounts ofenzyme and to purify this enzyme in a high yield.

Preferably, the recombinant diaminooxidase is expressed in prokaryotic,preferably in bacterial, or in eukaryotic, preferably in animal or yeastcell cultures and isolated from the expression systems indicated above.Purification of DAO produced by means of these expression systems, whichis either expressed in the cells or is secreted from the cells duringthe expression, is effected by methods known in the prior art. In doingso, it is also possible to provide the DAO with a peptide (e.g.His-tag), polypeptide or protein sequence (e.g. GST-tag) so as tosimplify said purification. The recombinant DAO may furthermore bemodified by genetic engineering methods such that the enzyme activity ofthis DAO surpasses the enzyme activity of the wild-type DAO.

A further aspect of the present invention relates to the use of thediaminoxidase according to the invention for producing a medicament forthe treatment of histamine-induced clinical pictures. DAO is known to beresponsible for the degradation of histamine in the human and animalbody. Since histamine-induced diseases are caused by an excess ofhistamine which is due to a lack of diaminooxidase or to the inhibitionof DAO, or by a histamine excess which, as a rule, may be caused by foodor also by further extrinsic factors, such as, e.g., contact withallergens, the administration of DAO of the invention lends itself tothe treatment of these diseases, or clinical pictures, respectively.

A further aspect of the present invention relates to the use of thediaminooxidase of the invention for producing a medicament for thetreatment of urticaria, in particular of chronic and acute urticaria.With urticaria, a release of histamine causes a widening of venoles andan excessive permeability of the capillaries with a resultant oedema. Byadministering DAO to the affected skin zones it is possible to degradethe histamine at the affected sites and to thereby stop the itching ofthe urticaria.

A further aspect of the present invention relates to the use ofdiaminooxidase of the invention for producing a medicament for thetreatment of contact allergies. Contact allergies are caused bysubstances (allergens) which trigger allergic reactions by penetratinginto the skin. In order to degrade the histamines released by thiscontact and thus, stop, or alleviate, respectively, thehistamine-induced clinical pictures, DAO is administered.

According to a further aspect of the present invention, a diaminooxidaseaccording to the invention is used for producing a medicament for thetreatment of atopic dermatitis. Atopic dermatitis, also known by thename neurodermatitis, is a frequent skin disease associated withpronounced itching, occurring mostly in children and in young adults.The cause of this itching is the excessive release of histamines at theaffected sites of the skin. Also in this case, DAO can attribute to thereduction of histamines in these areas and, thus, alleviate, or prevent,respectively, the symptoms of atopic dermatitis.

According to a further aspect, the present invention relates to the useof the diaminooxidase of the invention for producing a medicament forthe treatment of scombrotoxism. Scombrotoxism is a histamine poisoningafter the consumption of mackerel varieties, e.g. of tuna. Wheninterrupting the cold chain, or when delaying preparation, so-calledscombrotoxins form in scombrides, which lead to a histamine enrichment.The consequences of this histamine poisoning are i.a. fever, nausea,vomiting, bellyache, and urticaria. In this case, DAO can be used as adetoxicating agent.

A further aspect of the present invention relates to the use of thediaminooxidase of the invention for producing a medicament or a foodsupplement, or for a dietary foodstuff, for removing histamine from thegastro-intestinal tract, or reducing it therein, respectively.

The reduction of histamine in the intestinal tract is of particularimportance in case of an increased supply of histamine (exogenic supply,e.g. by food) to the body of an individual, or if, e.g., the activity ofthe DAO in the intestinal tract is partially or entirely inhibited, ornot provided at all, respectively. The medicament, or food supplement,or dietary foodstuff, respectively, of the invention thus serves forsupplying enzymatically active DAO which contributes to the degradationof histamine in the intestinal tract.

A further aspect of the present invention relates to the use ofdiaminooxidase for producing a medicament for removing histamine fromthe bronchial system.

The intake of histamine-releasing substances, such as, e.g., pollen,into the bronchial system, or into the lungs, respectively, can lead topronounced allergic reactions. In order to degrade the releasedhistamine, the DAO of the invention may, e.g., be introduced into thebronchial system, or into the lungs, respectively, by inhalation spraysand the like.

The invention will be further explained by the following exampleswithout, however, being restricted thereto.

EXAMPLES Example 1

DAO was actively stabilized in a cellulose-based pellet, and the pelletwas coated with a gastric-juice-resistant coating. In the dissolutiontest, it could be shown that more than 70% of the activity are releasedinto the surroundings within the first hour.

Example 2

Furthermore, the enzyme was stabilized in a hydrogel. Storage at roomtemperature and at 37° C. did not show a decrease in the activity within4 months. Occurring vesicles and itching of the skin after stimulationwith histamine disappeared a few minutes after application of thehydrogel.

Example 3

DAO stabilized in the hydrogel was tested on a total of 13 persons. Byway of a questionnaire it was distinguished between stress by contactallergy (n=5), neurodermatitis/dermatoses (n=6) and insect bites (n=2).In all patients with contact allergies and in neurodermatitis patients,a positive effect was attested; in case of insect bites, 50% of thevolunteers could report a positive effect.

Positive Effect/ Stress Number of Volunteers Neurodermatitis/Dermatosis6/6 Contact allergy 5/5 Insect bite 1/2

The effect of the hydrogel occurred within the first 10 minutes, theeffect on an average lasting for more than one hour.

0-10 min 10-30 min 30-60 min >1 h >3 h Start of effect 11 1 Duration ofeffect 3 1 2 6

No volunteer reported uncomfortable effects in case of a repeatedapplication.

Two neurodermatitis patients stated that the hydrogel of the inventionwas more effective than a cortisone ointment used at that time.

Example 4

Gastric-juice-resistant pellets were prepared with 3% of DAO which hadan initial activity of 80,000 U/ml, 40% of microcrystalline cellulose,20% of sucrose, 22% of other binders, fillers and disintegrating agentsand with 15% of a gastric-juice-resistant coating. The release of DAOfrom the pellets was observed for a period of 180 minutes by means of anactivity assay. Here, in the solution in which the pellets had beendissolved, already after 10 minutes a DAO activity of 40%, after 30minutes an activity of 70%, after 60 minutes an activity of 80% andafter 180 minutes an activity of 100% of the DAO amount originallyemployed was detected. The activity measurement of the DAO was asdescribed in AT 411688, wherein, however, also other known methods couldvery well be employed for measuring the enzyme activity.

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1.-18. (canceled)
 19. A pharmaceutical composition for treating ahistamine-induced disease, said composition comprising an effectiveamount of diaminooxidase and being provided in a form selected from thegroup consisting of a hydrogel, a gastric-juice-resistant pellet, agastric-juice-resistant capsule and a tablet, for an applicationselected from the group consisting of epidermal, oral and peroraladministration.
 20. A pharmaceutical composition as set forth in claim19, wherein said composition is a food supplement composition and isprovided in a form selected from the group consisting of agastric-juice-resistant pellet, a gastric-juice-resistant capsule and atablet.
 21. A pharmaceutical composition as set forth in claim 19,wherein said composition is a dietary foodstuff and is provided in aform selected from the group consisting of a gastric-juice-resistantpellet, a gastric-juice-resistant capsule and a tablet.
 22. Apharmaceutical composition as set forth in claim 19, wherein saidcomposition is a cosmetic composition.
 23. A pharmaceutical compositionas set forth in claim 22 and provided in a form for a cosmeticapplication, such as a hydrogel.
 24. A pharmaceutical composition as setforth in claim 19, wherein said diaminooxidase is a diaminooxidase ofnon-plant origin.
 25. A pharmaceutical composition as set forth in claim24, wherein said diaminooxidase is of animal origin.
 26. Apharmaceutical composition as set forth in claim 25, wherein saiddiaminooxidase of animal origin is derived from porcine kidney.
 27. Apharmaceutical composition as set forth in claim 19, wherein saiddiaminooxidase is of recombinant origin.
 28. A pharmaceuticalcomposition as set forth in claim 27, wherein said diaminooxidase ofrecombinant origin is recovered from one of prokaryotic and eukaryoticcell cultures.
 29. A pharmaceutical composition as set forth in claim28, wherein said prokaryotic cell cultures are bacterial cell cultures.30. A pharmaceutical composition as set forth in claim 28, wherein saideukaryotic cell cultures are selected from the group consisting ofanimal and yeast cell cultures.
 31. A method of treating ahistamine-induced disease in a patient, comprising administering to saidpatient an effective amount of diaminooxidase, said diaminooxidase beingprovided in a form selected from the group consisting of a hydrogel, agastric-juice-resistant pellet, a gastric-juice-resistant capsule and atablet, for an application selected from the group consisting ofepidermal, oral and peroral administration.
 32. A method as set forth inclaim 31, wherein said histamine-induced disease is urticaria, such aschronic and acute urticaria.
 33. A method as set forth in claim 31,wherein said histamine-induced disease is a contact allergy and saiddiaminooxidase is provided as a hydrogel for epidermal application. 34.A method as set forth in claim 31, wherein said histamine-induceddisease is an atopic dermatitis.
 35. A method as set forth in claim 31,wherein said histamine-induced disease is scombrotoxism and saiddiaminooxidase is provided in a form selected from the group consistingof a gastric-juice-resistant pellet, a gastric-juice-resistant capsuleand a tablet, for an application selected from the group consisting oforal and peroral administration.
 36. A method as set forth in claim 31,wherein said diaminooxidase is provided in a form selected from thegroup consisting of a gastric-juice-resistant pellet, agastric-juice-resistant capsule and a tablet, for an applicationselected from the group consisting of oral and peroral administration soas to remove histamine from the gastro-intestinal tract of said patient.37. A method of treating a histamine-induced disease in a patient,comprising administering to said patient a an effective amount ofdiaminooxidase, said diaminooxidase being provided in a form selectedfrom drops and a spray, so as to remove histamine from the bronchialsystem of said patient.